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. 2009 Feb 15;418(1):61-71.
doi: 10.1042/BJ20081304.

The selenocysteine tRNA STAF-binding region is essential for adequate selenocysteine tRNA status, selenoprotein expression and early age survival of mice

Bradley A Carlson  1 , Ulrich Schweizer, Christine Perella, Rajeev K Shrimali, Lionel Feigenbaum, Liya Shen, Svetlana Speransky, Thomas Floss, Soon-Jeong Jeong, Jennifer Watts, Victoria Hoffmann, Gerald F Combs, Vadim N Gladyshev, Dolph L Hatfield
Affiliations

The selenocysteine tRNA STAF-binding region is essential for adequate selenocysteine tRNA status, selenoprotein expression and early age survival of mice

Bradley A Carlson et al. Biochem J. .

Abstract

STAF [Sec (selenocysteine) tRNA gene transcription activating factor] is a transcription activating factor for a number of RNA Pol III- and RNA Pol II-dependent genes including the Trsp [Sec tRNA gene], which in turn controls the expression of all selenoproteins. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined. We generated transgenic mice expressing the Trsp transgene lacking the STAF-binding site and made these mice dependent on the transgene for survival by removing the wild-type Trsp. The level of Sec tRNA was unaffected or slightly elevated in heart and testis, but reduced approximately 60% in liver and kidney, approximately 70% in lung and spleen and approximately 80% in brain and muscle compared with the corresponding organs in control mice. Moreover, the ratio of the two isoforms of Sec tRNA that differ by methylation at position 34 (Um34) was altered significantly, and the Um34-containing form was substantially reduced in all tissues examined. Selenoprotein expression in these animals was most affected in tissues in which the Sec tRNA levels were most severely reduced. Importantly, mice had a neurological phenotype strikingly similar to that of mice in which the selenoprotein P gene had been removed and their life span was substantially reduced. The results indicate that STAF influences selenoprotein expression by enhancing Trsp synthesis in an organ-specific manner and by controlling Sec tRNA modification in each tissue examined.

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Figures

Figure 1
Figure 1. Survival rates of TrsptAE−/−/Trsp−/− and control mice
Survival rates of transgenic mice carrying two (n = 60) or ten copies (n = 54) of the mutant transgene were measured and found to be significantly different from control mice (P < 0.0001, the log-rank test) as shown in the Figure. The median survival rate of both TrsptAE−/−/Trsp−/− mouse lines was determined to be 48 days in mice carrying two and 46 days for mice carrying ten copies of TrsptAE−/−. Survival analysis was performed using GraphPad Prism 4.0.
Figure 2
Figure 2. Northern-blot analysis of TrsptAE−/−/Trsp−/− and control mice
Bulk tRNA was isolated from the various tissues shown in the Figure, prepared for electrophoresis and electrophoresed on gels and the levels of tRNA[Ser]Sec in TrsptAE−/−/Trsp−/− mice carrying two (A) or ten copies (B) of mutant transgene relative to control mice were determined as described in the Experimental section. Levels of tRNA[Ser]Sec in tissues from mice carrying two copies of mutant transgene and the corresponding controls were carried out on three different mice from both mouse lines. The amounts of tRNA[Ser]Sec were averaged in control mice and assigned a value of 1.0, while the error bars from the three assays with mutant mice are shown (A). Levels of tRNA[Ser]Sec in tissues from mice carrying ten copies of mutant transgene were carried out on a single mouse and compared with the averaged levels from three control mice (B).
Figure 3
Figure 3. Metabolic 75Se-labelling of selenoproteins in TrsptAE−/−/Trsp−/− and control mice
Mice were injected with 75Se; after 48 h, proteins were extracted and electrophoresed and the gels were stained with Coomassie Brilliant Blue and then exposed to a PhosphorImager to detect 75Se-labelled proteins. Coomassie Brilliant Blue-stained total protein from each tissue served as a loading control wherein the amounts of proteins observed from the Coomassie Brilliant Blue-stained gels were virtually identical in the same tissue from both TrsptAE−/−/Trsp−/− and control animals (results not shown). (A) 75Se-labelling of selenoproteins. Labelled selenoproteins observed in each tissue examined are shown along with molecular mass markers on the left side of the liver panel and selenoproteins identified in previous studies [6,42] are shown on the right sides of the liver and plasma panels. (B) Quantification of band intensities relative to corresponding control proteins. Band intensities of each selenoprotein evaluated in control tissues were assigned a value of 1.0 and the relative intensities of the corresponding bands in TrsptAE−/−/Trsp−/− mice are shown. Band intensities were assessed by PhosphorImager analysis as given in the Experimental section.
Figure 4
Figure 4. Western-blot analysis of selenoproteins affected by reduction of the mcm5Um isoform
(A) Protein extracts were prepared from TrsptAE−/−/Trsp−/− and control mice in the tissues shown in the Figure, electrophoresed, transblotted on to membranes and treated with antibodies as detailed in the Experimental section. The blots shown were from tissues of a single mouse and are representative of the selenoproteins from the corresponding tissues of three different animals. GP×1 was not detected in muscle. Tubulin was used as a loading control. (B) Levels of analysed selenoproteins from the tissues of three mice relative to the corresponding selenoprotein levels from control tissues. The asterisk indicates that the differences in selenoprotein levels were significant (P < 0.05) between control and affected tissues as determined by the Student's t test (unpaired, two-sided) using GraphPad Prism 4.0 as described in the Experimental section.
Figure 5
Figure 5. Pathological analysis of spleen, brain and liver in TrsptAE−/−/Trsp−/− and control mice
Panels show H/E-stained tissues of control (left side) and TrsptAE−/−/Trsp−/− (right side) mice. (A) Spleens of six of eight TrsptAE−/−/Trsp−/− mice were smaller in size with smaller splenic follicles (black arrows show comparisons between control and mutant spleen), wherein the follicles also lacked a marginal zone (note the white arrow in the left panel showing marginal zone in control spleen), when compared with control mice. (B) Brains of five of eight TrsptAE−/−/Trsp−/− mice manifested acute neuronal necrosis characterized by hypereosinophilic shrunken neurons (marked by arrows) compared with control mice. (C) Livers of three of eight TrsptAE−/−/Trsp−/− mice had foci of necrosis (marked by arrow) and hepatocytes were smaller than in control mice.
Figure 6
Figure 6. Histological analysis of brain tissues from TrsptAE−/−/Trsp−/− and control mice
Analysis of control and TrsptAE−/−/Trsp−/− mice is shown in the left and right panels respectively. (A) Cresyl Violet (Nissl) staining of hippocampus (upper panel) and somatosensory cortex (lower panel). The arrow points to a region of diffuse neuronal death with a high fraction of pyknotic cells. (B) Immunohistochemistry of GFAP. Antibodies directed against GFAP revealed massive astrogliosis in hippocampus and cerebral cortex grey matter in TrsptAE−/−/Trsp−/− compared with control mice. (C) Immunohistochemistry for PV. Antibodies directed against PV demonstrated a decreased number of PV-immunopositive GABAergic interneurons in hippocampus and somatosensory cortex in TrsptAE−/−/Trsp−/− mice compared with control mice. N = 5 per genotype. Scale bar, 200 μm.
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